Revealing CO2-fixing SAR11 bacteria in the ocean by Raman-based single-cell metabolic profiling and genomics

The majority of marine microbes remain uncultured, which hinders the identification and mining of CO2-fixing genes, pathways, and chassis from the oceans. Here, we investigated CO2-fixing microbes in seawater from the euphotic zone of the Yellow Sea of China by detecting and tracking their 13C-bicarbonate (13C-HCO3-) intake via single-cell Raman spectra (SCRS) analysis. The target cells were then isolated by Raman-activated Gravity-driven Encapsulation (RAGE), and their genomes were amplified and sequenced at one-cell resolution. The single-cell metabolism, phenotype and genome are consistent. We identified a not-yet-cultured Pelagibacter spp., which actively assimilates 13C-HCO3-, and also possesses most of the genes encoding enzymes of the Calvin-Benson cycle for CO2 fixation, a complete gene set for a rhodopsin-based light-harvesting system, and the full genes necessary for carotenoid synthesis. The four proteorhodopsin (PR) genes identified in the Pelagibacter spp. were confirmed by heterologous expression in E. coli. These results suggest that hitherto uncultured Pelagibacter spp. uses light-powered metabolism to contribute to global carbon cycling

Genome engineering of Nannochloropsis with hundred-kilobase fragment deletions by Cas9 cleavages

Industrial microalgae are promising photosynthetic cell factories, yet tools for large-scale targeted genome engineering are limited. Here for the model industrial oleaginous microalga Nannochloropsis oceanica, we established a method to precisely and serially delete large genome fragments of similar to 100 kb from its 30.01 Mb nuclear genome. We started by identifying the 'non-essential' chromosomal regions (i.e. low expression region or LER) based on minimal gene expression under N-replete and N-depleted conditions. The largest such LER (LER1) is similar to 98 kb in size, located near the telomere of the 502.09-kb-long Chromosome 30 (Chr 30). We deleted 81 kb and further distal and proximal deletions of up to 110 kb (21.9% of Chr 30) in LER1 by dual targeting the boundaries with the episome-based CRISPR/Cas9 system. The telomere-deletion mutants showed normal telomeres consisting of CCCTAA repeats, revealing telomere regeneration capability after losing the distal part of Chr 30. Interestingly, the deletions caused no significant alteration in growth, lipid production or photosynthesis (transcript-abundance change for < 3% genes under N depletion). We also achieved double-deletion of both LER1 and LER2 (from Chr 9) that total similar to 214 kb at maximum, which can result in slightly higher growth rate and biomass productivity than the wild-type. Therefore, loss of the large, yet 'non-essential' regions does not necessarily sacrifice important traits. Such serial targeted deletions of large genomic regions had not been previously reported in microalgae, and will accelerate crafting minimal genomes as chassis for photosynthetic production

Integration of proteome and transcriptome refines key molecular processes underlying oil production in Nannochloropsis oceanica\textit {Nannochloropsis oceanica}

$\bf Background$ Under nitrogen deficiency situation, Nannochloropsis spp. accumulate large amounts of lipids in the form of triacylglycerides (TAG). Mechanisms of this process from the perspective of transcriptome and metabolome have been obtained previously, yet proteome analysis is still sparse which hinders the analysis of dynamic adaption to nitrogen deficiency. Here, proteomes for 3 h, 6 h, 12 h, 24 h, 48 h and 10th day of nitrogen deplete (N−) and replete (N+) conditions were obtained and integrated with previous transcriptome data for $\textit {N. oceanica}$. $\bf Results$ Physiological adaptations to N− not apparent from transcriptome data were unveiled: (a) abundance of proteins related to photosynthesis only slightly decreased in the first 48 h, indicating that photosynthesis is still working efficiently, and protein amounts adjust gradually with reduction in chloroplast size. (b) Most proteins related to the TCA cycle were strongly upregulated after 48 h under N−, suggesting that respiration is enhanced after 48 h and that TCA cycle efflux supports the carbon required for lipid synthesis. (c) Proteins related to lipid accumulation via the Kennedy pathway increased their abundance at 48 h, synchronous with the previously reported diversification of fatty acids after 48 h. $\bf Conclusions$ This study adds a proteome perspective on the major pathways for TAG accumulation in $\textit {Nannochloropsis spp}$. Temporal changes of proteome exhibited distinct adaptation phases that are usually delayed relative to transcriptomic responses. Notably, proteome data revealed that photosynthesis and carbon fixation are still ongoing even after 48 h of N−. Moreover, sometimes completely opposite trends in proteome and transcriptome demonstrate the relevance of underexplored post-transcriptional regulation for N− adaptation

Single-cell Raman-activated sorting and cultivation (scRACS-Culture) for assessing and mining in situ phosphate-solubilizing microbes from nature

Abstract Due to the challenges in detecting in situ activity and cultivating the not-yet-cultured, functional assessment and mining of living microbes from nature has typically followed a ‘culture-first’ paradigm. Here, employing phosphate-solubilizing microbes (PSM) as model, we introduce a ‘screen-first’ strategy that is underpinned by a precisely one-cell-resolution, complete workflow of single-cell Raman-activated Sorting and Cultivation (scRACS-Culture). Directly from domestic sewage, individual cells were screened for in-situ organic-phosphate-solubilizing activity via D2O intake rate, sorted by the function via Raman-activated Gravity-driven Encapsulation (RAGE), and then cultivated from precisely one cell. By scRACS-Culture, pure cultures of strong organic PSM including Comamonas spp., Acinetobacter spp., Enterobacter spp. and Citrobacter spp., were derived, whose phosphate-solubilizing activities in situ are 90–200% higher than in pure culture, underscoring the importance of ‘screen-first’ strategy. Moreover, employing scRACS-Seq for post-RACS cells that remain uncultured, we discovered a previously unknown, low-abundance, strong organic-PSM of Cutibacterium spp. that employs secretary metallophosphoesterase (MPP), cell-wall-anchored 5′-nucleotidase (encoded by ushA) and periplasmic-membrane located PstSCAB-PhoU transporter system for efficient solubilization and scavenging of extracellular phosphate in sewage. Therefore, scRACS-Culture and scRACS-Seq provide an in situ function-based, ‘screen-first’ approach for assessing and mining microbes directly from the environment

Comparative analysis of the gut microbiota composition between knee osteoarthritis and Kashin-Beck disease in Northwest China

Background: Osteoarthritis (OA) and Kashin-Beck disease (KBD) both are two severe osteochondral disorders. In this study, we aimed to compare the gut microbiota structure between OA and KBD patients. Methods: Fecal samples collected from OA and KBD patients were used to characterize the gut microbiota using 16S rDNA gene sequencing. To identify whether gut microbial changes at the species level are associated with the genes or functions of the gut bacteria between OA and KBD groups, metagenomic sequencing of fecal samples from OA and KBD subjects was performed. Results: The OA group was characterized by elevated Epsilonbacteraeota and Firmicutes levels. A total of 52 genera were identified to be significantly differentially abundant between the two groups. The genera Raoultella, Citrobacter, Flavonifractor, g__Lachnospiraceae_UCG-004, and Burkholderia-Caballeronia-Paraburkholderia were more abundant in the OA group. The KBD group was characterized by higher Prevotella_9, Lactobacillus, Coprococcus_2, Senegalimassilia, and Holdemanella. The metagenomic sequencing showed that the Subdoligranulum_sp._APC924/74, Streptococcus_parasanguinis, and Streptococcus_salivarius were significantly increased in abundance in the OA group compared to those in the KBD group, and the species Prevotella_copri, Prevotella_sp._CAG:386, and Prevotella_stercorea were significantly decreased in abundance in the OA group compared to those in the KBD group by using metagenomic sequencing. Conclusion: Our study provides a comprehensive landscape of the gut microbiota between OA and KBD patients and provides clues for better understanding the mechanisms underlying the pathogenesis of OA and KBD

Genetic Variants and Protein Alterations of Selenium- and T-2 Toxin-Responsive Genes Are Associated With Chondrocytic Damage in Endemic Osteoarthropathy

The mechanism of environmental factors in Kashin-Beck disease (KBD) remains unknown. We aimed to identify single nucleotide polymorphisms (SNPs) and protein alterations of selenium- and T-2 toxin-responsive genes to provide new evidence of chondrocytic damage in KBD. This study sampled the cubital venous blood of 258 subjects including 129 sex-matched KBD patients and 129 healthy controls for SNP detection. We applied an additive model, a dominant model, and a recessive model to identify significant SNPs. We then used the Comparative Toxicogenomics Database (CTD) to select selenium- and T-2 toxin-responsive genes with the candidate SNP loci. Finally, immunohistochemistry was applied to verify the protein expression of candidate genes in knee cartilage obtained from 15 subjects including 5 KBD, 5 osteoarthritis (OA), and 5 healthy controls. Forty-nine SNPs were genotyped in the current study. The C allele of rs6494629 was less frequent in KBD than in the controls (OR = 0.63, p = 0.011). Based on the CTD database, PPARG, ADAM12, IL6, SMAD3, and TIMP2 were identified to interact with selenium, sodium selenite, and T-2 toxin. KBD was found to be significantly associated with rs12629751 of PPARG (additive model: OR = 0.46, p = 0.012; dominant model: OR = 0.45, p = 0.049; recessive model: OR = 0.18, p = 0.018), rs1871054 of ADAM12 (dominant model: OR = 2.19, p = 0.022), rs1800796 of IL6 (dominant model: OR = 0.30, p = 0.003), rs6494629 of SMAD3 (additive model: OR = 0.65, p = 0.019; dominant model: OR = 0.52, p = 0.012), and rs4789936 of TIMP2 (recessive model: OR = 5.90, p = 0.024). Immunohistochemistry verified significantly upregulated PPARG, ADAM12, SMAD3, and TIMP2 in KBD compared with OA and normal controls (p < 0.05). Genetic polymorphisms of PPARG, ADAM12, SMAD3, and TIMP2 may contribute to the risk of KBD. These genes could promote the pathogenesis of KBD by disturbing ECM homeostasis

Single‐cell rapid identification, in situ viability and vitality profiling, and genome‐based source‐tracking for probiotics products

Abstract Rapid expansion of the probiotics industry demands fast, sensitive, comprehensive, and low‐cost strategies for quality assessment. Here, we introduce a culture‐free, one‐cell‐resolution, phenome‐genome‐combined strategy called Single‐Cell Identification, Viability and Vitality tests, and Source‐tracking (SCIVVS). For each cell directly extracted from the product, the fingerprint region of D2O‐probed single‐cell Raman spectrum (SCRS) enables species‐level identification with 93% accuracy, based on a reference SCRS database from 21 statutory probiotic species, whereas the C–D band accurately quantifies viability, metabolic vitality plus their intercellular heterogeneity. For source‐tracking, single‐cell Raman‐activated Cell Sorting and Sequencing can proceed, producing indexed, precisely one‐cell‐based genome assemblies that can reach ~99.40% genome‐wide coverage. Finally, we validated an integrated SCIVVS workflow with automated SCRS acquisition where the whole process except sequencing takes just 5 h. As it is >20‐fold faster, >10‐time cheaper, vitality‐revealing, heterogeneity‐resolving, and automation‐prone, SCIVVS is a new technological and data framework for quality assessment of live‐cell products

The NanDeSyn database for Nannochloropsis systems and synthetic biology

Nannochloropsis species, unicellular industrial oleaginous microalgae, are model organisms for microalgal systems and synthetic biology. To facilitate community-based annotation and mining of the rapidly accumulating functional genomics resources, we have initiated an international consortium and present a comprehensive multi-omics resource database named Nannochloropsis Design and Synthesis (NanDeSyn; ). Via the Tripal toolkit, it features user-friendly interfaces hosting genomic resources with gene annotations and transcriptomic and proteomic data for six Nannochloropsis species, including two updated genomes of Nannochloropsis oceanica IMET1 and Nannochloropsis salina CCMP1776. Toolboxes for search, Blast, synteny view, enrichment analysis, metabolic pathway analysis, a genome browser, etc. are also included. In addition, functional validation of genes is indicated based on phenotypes of mutants and relevant bibliography. Furthermore, epigenomic resources are also incorporated, especially for sequencing of small RNAs including microRNAs and circular RNAs. Such comprehensive and integrated landscapes of Nannochloropsis genomics and epigenomics will promote and accelerate community efforts in systems and synthetic biology of these industrially important microalgae